Quality assurance/quality control for electrospray ionization processes

ABSTRACT

The present invention relates to a method of quality assurance/quality control for high-throughput bioassay processes. The method includes generating a bioassay process model, and then comparing spectral data based on a combination of a biochip and a test serum to the bioassay process model to determine if the test sample and the bioassay process are producing acceptable data. Alternatively, the method may include comparing spectral data based on a combination of serum and diluents used in an electrospray process to the bioassay process model. If the bioassay process and test sample fall within the model, then the spectrum produced may be further analyzed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 60/398,831, entitled “QualityAssurance/Quality Control for SELDI-TOF Mass Spectra,” filed on Jul. 29,2002, the contents of which are hereby incorporated by reference intheir entirety.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

The research work described here was supported under a CooperativeResearch and Development Agreement (CRADA) between the US Government andCorrelogic Systems, Inc.

BACKGROUND OF THE INVENTION

The present invention relates generally to the field of bioinformatics.More specifically, the present invention relates to a method of qualityassurance/quality control (“QA/QC”) for bioinformatic systems.

Methods of analyzing biological samples are generally known. In atypical analysis, mass spectroscopy is performed on the biologicalsample to determine its overall biochemical make-up. Based on the massspectra obtained from the mass spectroscopy, various diagnostics may berun.

When biological samples are analyzed, it is desirable to run more thanone trial on the biological sample, thereby improving the accuracy ofthe diagnostic. Analysis of biological samples may be performed by usingvarious high-throughput mass spectrometry related bioassay processes. Aprocess can include using matrix assisted laser desorption ionizationtime-of-flight (MALDI-TOF) or electrospray techniques (i.e., generationof droplets by applying a high voltage to a stream of liquid). Whenperforming multiple mass spectral analyses on the same sample, however,the spectra obtained can vary. This variation may be due to the massspectrometer itself, from inconsistencies in the sample, heterogeneityin the patient population, or in sample handling and processing. Aprocess that employed a protein chip or surface enhanced type of massspectrometry (SELDI-TOF) indicated that various chips yielded spectrathat were inconsistent with one another. Similar effects were observedwith respect to spectra obtained using electrospray techniques. Thisinconsistency can lead to inaccurate results when running a diagnostic.

SUMMARY OF THE INVENTION

The present invention provides a QA/QC method for filtering outinconsistencies across high-throughput bioassay processes, particularlyacross different biochips and different diluents or concentrations ofdiluents used in electrospray techniques.

The present invention uses the Knowledge Discovery Engine (“KDE”) toidentify hidden patterns across a wide variety of serum samples andbiochips to generate a control model and agnostic to the underlyingdisease processes in question. Electrospray, MALDI-TOF (Matrix AssistedLaser Desorption/Ionization-Time of Flight) mass spectra, or SELDI-TOF(Surface Enhanced Laser Desorption/Ionization-Time of Flight) massspectra can be analyzed in this manner, for example. Alternatively, theinvention may use the KDE to identify hidden patterns across a varietyof serum to diluent concentrations to generate a control model. In yetanother embodiment, the KDE may be used to identify hidden patternsacross a variety of diluents and sera samples to generate a controlmodel.

The KDE is disclosed in U.S. patent application Ser. No. 09/883,196, nowU.S. Pat. No. 7,096,206, entitled “Heuristic Methods of Classification,”filed Jun. 19, 2001 (“Heuristic Methods”), and U.S. patent applicationSer. No. 09/906,661, now U.S. Pat. No. 6,925,389 entitled “A Process forDiscriminating Between Biological States Based on Hidden Patterns fromBiological Data,” filed Jul. 18, 2001 (“Hidden Patterns”); the contentsof both applications are hereby incorporated by reference in theirentirety. Software running the KDE is available from Correlogic Systems,Inc., under the name Proteome Quest™.

After the KDE is used to generate a control model, a test serum may becompared to the control model to determine if the spectra produced bythe high-throughput bioassay process and the serum are acceptable.

The KDE will search for hidden or subtle patterns of molecularexpression that are, in and of themselves, “diagnostic.” The level ofthe identified molecular products is termed per se diagnostic, becausethe level of the product is diagnostic without any further considerationof the level of any other molecular products in the sample.

In the data cluster analysis utilizing the KDE, the diagnosticsignificance of the level of any particular marker, e.g., a protein ortranscript, is a function of the levels of the other elements that areused to calculate a sample vector. Such products are referred to as“contextual diagnostic products.” The KDE's learning algorithm discoverswholly new classification patterns without knowing any prior informationabout the identity or relationships of the data pattern, i.e., withoutprior input that a specified diagnostic molecular product is indicativeof a particular classification.

If the spectrum produced by the biochip and the serum map to the controlmodel, then the data obtained from mass spectrometry of the serum andbiochip may be used for further analysis. If the spectrum produced bythe biochip and the serum fail to map to the control model, the data isdeemed uncertified, and new data must be acquired. Alternatively, if aspectrum produced by a serum sample and a diluent map to the controlmodel, then the spectrum obtained from an electrospray process may beused for further analysis. By using this method, inconsistencies acrossbioassay processes may be avoided, thereby improving the reliability ofdata obtained using the bioassay process. Other advantages may also berealized from the methods disclosed herein, as would be obvious to theordinarily skilled artisan.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart illustrating a method of obtaining a controlmodel.

FIG. 2 depicts a table having various serum/biochip combinations thatmay be used to obtain the control model.

FIG. 3 illustrates a method of comparing the test serum to the controlmodel.

FIG. 4 is a depiction of mapping of exemplary tolerances inthree-dimensional space according to one aspect of the presentinvention.

FIG. 5 is a table illustrating results obtained from the KDE using twodifferent types of biochips and 256 sera.

FIG. 6 is a flow chart of an alternative embodiment of the presentinvention for use with an electrospray process.

DETAILED DESCRIPTION

Generally, the invention includes a method of obtaining a control modelfor use in a bioinformatics system and a method for comparing a testsample against the model for the purpose of QA/QC.

A method of obtaining a control model according to one aspect of thepresent invention is illustrated in FIG. 1. To ensure a highlyarticulate model, a variety of serum samples are selected at step 100.The selection should include selecting serum from as diverse a group ofindividuals as possible. The more diverse the selected sera, the morearticulate the control model will be. For example, sera may be takenfrom healthy males, healthy females, males afflicted with variousdiseases, females afflicted with various diseases, persons of differentages, and persons of different races.

Once the diverse group of sera has been selected, a group of differentbiochips is selected at step 110. The diverse group of biochips mayinclude an anionic chip, a cationic chip, and an immobilized metal chip.The selection of chips may include at least one anionic chip and atleast one cationic chip. However, in order to generate a workable modelat least two chips should be selected. For example, one model could begenerated using three types of chips: WCX2 (cationic exchange), SAX2(anionic exchange), and IMAC3 (immobilized metal) surface enhanced laserdesorption/ionization (“SELDI”) chips.

After the initial selection of sera (100) and the selection of biochips(110), the sera are applied to the chips in step 120. After each serumis applied to the surface of a chip, then each chip and sera combinationis analyzed by mass spectrometry at step 130 to obtain a spectral outputcharacterized by mass to charge (m/z) values. After one spectrum isproduced, the process is repeated for a different biochip/serumcombination. Each time a spectrum is obtained for a particularbiochip/serum combination, a determination is made at step 140 ofwhether all chips have been analyzed.

After all of the chips have been analyzed, a determination is made atstep 150 of whether all sera have been analyzed by mass spectrometry incombination with each chip type. For example, assume three sera areselected at step 100, and two biochips are selected (one cationicexchange biochip (“Biochip A”) and one anionic exchange biochip(“Biochip B”)). After the first serum is analyzed by mass spectrometryon Biochip A, a determination is made at step 140 of whether allbiochips have been used. Finding that the first serum has not been usedwith Biochip B, the process is repeated starting with step 120.

When both Biochip A and Biochip B have been analyzed with the firstserum, a determination is made at step 150 of whether there are any moresera remaining. If any more sera remain, the process is repeated foreach of the biochips. In this example, the process will be repeated foreach of Biochip A and Biochip B, with the second and third serarespectively.

The data for each of the spectra may be stored, such as in the tableillustrated in FIG. 2. The table includes data for “i” sera and “j”chips. Each cell in the table contains mass spectra (MS) data associatedwith each chip type and the various types of serum. For example, cellMS_(j,i) corresponds to the spectral data from chip “j” and serum sample“i”. After all of the data have been obtained, the stored mass spectrumdata can be input into the KDE to discover hidden patterns in thespectral data.

Next, the process of developing a biochip model using the KDE will bedescribed.

The data from each of the mass spectra are input into the KDE. The KDEthen seeks to identify clusters of data (hidden patterns) inn-dimensional space, where n is the number of mass to charge valuesselected from the spectra for analysis, and each spectrum can be mappedinto the n-dimensional space using the magnitude of each of the selectedmass to charge values in the spectrum (the combination of a mass tocharge value and the magnitude of the spectrum at that value being avector). The KDE seeks clusters that contain as many of the spectra aspossible and that distinguish each of the biochips from the others. Eachcluster of data will define a centroid that will be associated with aparticular biochip. In the event that a number of possible groupings orcombinations of clusters are identified by the KDE, the user will selectthe most optimal grouping to define the biochip model. The selectionprocess could be automated rather than being directly performed by theuser. In either case, the cluster with the highest population of vectorscan be identified by either the user or the system and that cluster canbe designated as the control model.

After the model has been obtained, test sera may be run against themodel to determine if the sera/biochip combination is certified forfurther analysis. One method of QA/QC using the biochip model obtainedin FIG. 1 is depicted in FIG. 3.

First, in step 300 multiple samples from a test serum is applied to abiochip. The test serum could be serum intended for a cancer screening,for example. Then in step 310 the test serum samples are analyzed bymass spectrometry. The spectra obtained in step 310 are then mapped tothe biochip model in step 320.

Mapping the spectrum to the biochip model is performed in manner similarto the mapping of a serum sample to a training data set to diagnose aparticular disease state as described in the Hidden Patternsapplication. Mapping a spectrum to the biochip model includesdetermining the spectrum value for each of the n mass to charge ratiosin the biochip mode, plotting the associated vectors in the model'sn-dimensional space, and comparing them with the location of the model'scentroid. The centroid is defined as the center of the clusterdetermined to have the highest population of vectors from the modeldevelopment.

After the spectrum of the test sample is mapped to the biochip model, itis determined in a step 330 if the spectrum maps within a predeterminedhypervolume centered on the centroid in the model associated with thetested biochip.

If the spectrum maps within the predetermined hypervolume, the spectrumis deemed certified for further analysis. If the map of the spectrumfalls outside the predetermined hypervolume, the spectrum is not deemedcertified and the sample must be reanalyzed.

A system employing the method of the present invention should operatewithin predetermined tolerances. In determining whether a spectrum for asample maps to the model for the biochip used with the sample, the pointto which the vectors from each sample spectrum maps in the model'sn-dimensional space maps are compared to the location of the centroidfor the cluster associated with the selected biochip. The spectrum isconsidered to map to the model if it lies within a predeterminedhypervolume centered on that centroid. In this embodiment, thehypervolume defined with the centroid as its center will excludeapproximately 95% of the total hypervolume of the n-dimensional space.The content of a polytope or other n-dimensional object is itsgeneralized volume (i.e., its “hypervolume”). Just as athree-dimensional object has volume, surface area and generalizeddiameter, an n-dimensional object has “measures” of order 1, 2, . . . ,n, the hypervolume is defined based on these measures of order. Thehypervolume can also be defined in terms of a predefined acceptableprocess tolerance. The n-dimensional hypervolume calculation is akin toMahalanobis distances used in establishing rejection and acceptancecriteria.

This can be visualized in three dimensions as depicted in FIG. 4. FIG. 4illustrates a centroid “C,” which is associated with the cluster offeatures plotted in n-dimensional space (here the space is depicted asthree-dimensional for visualization purposes). A theoretical sphere, “S”is located in the n-dimensional space. The sphere is centered at thelocation of the centroid “C.” Tolerances should be set such that thesphere “S” has a volume that excludes approximately 95% of the volume ofthe three-dimensional space. The volume of the three dimensional spaceis defined by the set of plotted features in that space, and is boundedby (and preferably normalized to) the m/z feature with the greatestintensity.

Referring back to FIG. 3, once a sample is deemed certified, thespectral data may be used for further analysis. The further analysis mayentail running the data through the KDE to discover hidden patterns inthe spectra. These hidden patterns may be compared to disease statemodels to diagnose for a particular disease. This method of diagnosis isdescribed in further detail in the Hidden Patterns application.

If no spectra map to the model, then in step 350 the sample and biochipare deemed non-certified. If the sample and the chip were deemednon-certified the for the first time using a particular serum sample,then in steps 360 and 370 a determination is made to see if the “nocertification” is a first “no certification”; is so, a new chip isselected and the process repeated.

If the sample and the biochip were determined to be non-certified morethan once, then in step 380 a new serum sample and chip are obtained,and the process is repeated. After a certified sample has been obtained,at step 410 it is output for further analysis.

In one exemplary test, sera was obtained from subjects with and withoutcancer. Two types of biochips were selected, IMAC3 (immobilized metal)and WCX2 (cationic exchange). A diverse group of 256 sera were selected.The sera were then applied to each type of chip, analyzed by massspectrometry, and the spectral output collected. The spectral data wasinput into the KDE.

The model identified by the KDE is shown in FIG. 5. The table in FIG. 5shows the constituent “patterns” or clusters comprising the model. Eachcluster corresponds to a point, or node, in the N-dimensional spacedefined by the N m/z values (or “features”) included in the model. Inthis case, 10 m/z values are included in the model, so n=10. The tableshows the constituent centroids of the mode, each in a row identified bya “Node” number. Thus, this model has eight nodes or centroids. Thetable also includes columns for the constituent features or vectors ofthe centroids, with the m/z value for each vector identified at the topof the column. The amplitudes are shown for each feature or m/z value,for each centroid, and are normalized to 1.0.

The remaining four columns in the table are labeled “Count,” “State,”“StateSum,” and “Error.” “Count” is the number of samples in theTraining set that correspond to the identified node. “State” indicatesthe state of the node, where 1 indicates the IMAC3 chip and 0 indicatesthe WCX2 chip. “StateSum” is the sum of the state values for all of thecorrectly classified members of the indicated node, while “Error” is thenumber of incorrectly classified members of the indicated node. Thus,for node 2, 108 samples were assigned to the node, whereas 104 sampleswere actually from the IMAC3 chip. StateSum is thus 104 (rather than108) and Error is 4.

The cluster that contained the highest population of vectors wasdesignated as the control model. In the Example illustrated in FIG. 5,node 2 defined the control model because it contained the greatestnumber of vectors (108).

While the method described above has been described for use withbiochips, another embodiment of the invention may be practiced usingelectrospray techniques to obtain data relating to a particular serum.

When using an electrospray technique for obtaining biological data fordiagnostic purposes, a primary factor limiting the consistency of thedata obtained is the particular diluents used in preparing the samplefor electrospray analysis. Therefore, rather than characterizing theserum in conjunction with the biochips being used, electrospraytechniques used in combination with the present methods willcharacterize the serum in combination with the diluents used.

To use electrospray ionization (“ESI”) to obtain accurate spectral data,a stable spray should be obtained. There are three physicalcharacteristics of the spectral data that yield spectral results thatcan be utilized by the KDE. These physical characteristics include: (1)the number of mass peaks; (2) the total ion current; and (3) thestability of the spray.

Various tests were run to determine a preferred diluent concentrationand composition to yield effective results for use in the KDE. Thesetests were run using electrospray apparatus manufactured by AdvionBioSciences, and particularly the NanoMate100™ ESI in conjunction withCorrelogic's Proteome Pattern Blood Test™ which is based on the ProteomeQuest™ software.

One particular test involved diluting the serum sample at 1:1000 in50:50 acetonitrile: H₂O containing 0.2% formic acid (FA). While 20 λ ofeach sample was aliquoted into each well, representing 0.02 λ of serum,only about 100-200 nano-liters was actually sprayed and analyzed.Operating the Q Star mass spectrometer in positive ion TOF/MS mode, datawas acquired for 2 minutes/sample, m/z of 300-2000, using a 1 secondscan rate. The nanospray was initiated by applying 1.55 kV spray voltageat a pressure of about 0.5 psi.

Based on testing of serum samples a determination was made that theMultichannel acquisition mode (MCA) should be used on the Q-star massspectrometer when obtaining spectral data. This is based on adetermination that the MCA mode produced better resolution of thespectral peaks.

To optimize the diluent used in preparing the serum samples, variousother tests were run. The concentrations of the serum to diluent arepreferably between 1:1000 and 1:250, but other diluent concentrationsmay be selected in a manner apparent to those skilled in the art. Twodiluent types tested included acetonitrile (ACN) and methanol (MeOH).Each of these diluents was combined with an acid. Acids include, but arenot limited to, trifluoroacetic acid (TFA), formic acid (FA), and aceticacid. Either TFA or FA can be used for purposes of the present inventionand are preferably in concentrations between about 0.2% acid and 1.0%acid.

An alternative embodiment of the method of obtaining a model accordingto the present invention will now be described in relation to FIG. 6.The step of selecting sera in step 100 is the same as described above.Again, the more diverse the overall group of selected sera is, the morearticulate the model will be.

Secondly, in step 610 a selection of diluents is made. Diluents selectedmay be diverse or homogeneous. For example, a diverse group of diluentsincluding ACN and MeOH may be selected. Alternatively, only ACN may beselected as long as the concentrations of the ACN differ (e.g., 1:1000,1:500, and 1:250 serum to ACN).

Thirdly, in step 620, a mixture is made according to predeterminedconcentrations of serum to diluent. This mixture is then analyzed usingelectrospray in step 630 to obtain spectral data. This process isrepeated for all desired concentrations, diluents and serum samplesuntil all data have been obtained in steps 640, 650, and 660. A model isthen obtained in step 670 based on the data extracted from the varioussamples. The model is obtained using the KDE in the same manner asdescribed above for the acquisition of a biochip model.

A method of QA/QC using the electrospray is substantially the same asfor the disclosed method of QA/QC for biochips. Notable variations canbe found in the generation of the sample and the method of obtaining thedata. The significant differences between the overall processes stemsfrom the differences in obtaining the model and its ability to identifya particular serum diluent.

In one particular test, sera were obtained from male and femalesubjects. Two diluents, acetonitrile (ACN) and methanol (MeOH) wereselected. A mixture was made at a concentration of 1:250 of serum todiluent. Selected sera mixture (102 samples) were analyzed byelectrospray mass spectrometry, and the spectral output collected. Thespectral data was input into the KDE. The KDE identified a modelcontaining three clusters in total that distinguished the two diluents.One cluster was associated with ACN and that cluster was designated asthe control model.

The above-described method is applicable to various bioassay processesto ensure that both the particular high-throughput bioassay processbeing used and the serum being tested will yield an accurate diagnostic.By using the method described above, biological diagnostics may beprovided that have increased accuracy and reliability.

Tolerances to employ the aforementioned methods were described as beingsuch that a hypervolume defined about the centroid of a cluster thatwill exclude approximately 95% of the total hypervolume of then-dimensional space. While 95% percent was explicitly mentioned, one ofordinary skill in the art would realize that the methods of the presentinvention would operate effectively with different sized hypervolumescentered on the centroid.

In the described embodiments, biochips and electrospray processes wereillustrative. Various other high-throughput bioassay processes are knownin the art and could be employed with the methods of the presentinvention.

In the described embodiments, to obtain a model characteristic of aparticular high-throughput bioassay process, sera should be taken fromhealthy males, healthy females, males afflicted with a disease, femalesafflicted with a disease, persons of different ages and persons ofdifferent races. While these specific examples were given, numerousother diverse sera samples could be taken. The best possible diversesera would contain serum from every individual in the world. Therefore,taking sera from any individual that does not group into one of theaforementioned classifications is within the scope of the presentinvention.

While specific diluents and acids were described in reference to themethods of QA/QC for electrospray techniques, these diluents and acidsare not intended to be exhaustive and a variety of other suitablediluents and acids are suitable for those explicitly mentioned.Additionally, while specific concentrations of both acids and ratios ofsera to diluent were disclosed, one of ordinary skill in the art willrealize the specific concentrations will depend on the particular acidsand diluents used to perform the inventive method, and the describedacids and diluents are not intended to be all inclusive. Various otherconcentrations in combination with various acids and diluents will beobvious to the ordinarily skilled artisan based on the teachings of thepresent invention.

The various features of the invention have been described in relation toa method of quality assurance/quality control of high-throughputbioassay processes. However, it will be appreciated that many of thesteps may be implemented with various apparatus and bioinformaticsmethods. Moreover, variations and modifications exist that would notdepart from the scope of the invention.

1. A method of determining whether mass spectral data from a test serumis acceptable for analysis in a bioassay, comprising: selecting adiverse group of sera, the diverse group of sera having differentcharacteristics; diluting each serum of the diverse group of sera with aplurality of different diluents; obtaining information associated with amass spectrum of each of the diluted sera from the diverse group of serausing an electrospray process; generating a control model based at leastin part on the spectra obtained from the diverse group of sera, thecontrol model including at least one centroid located in ann-dimensional space defined by n mass spectral features included in thecontrol model; diluting a test serum with a test diluent; performingmass spectrometry on the test serum to obtain a test spectrum associatedwith the test serum; mapping the test spectrum to the n-dimensionalspace; and certifying that the test spectrum is acceptable for analysisin the bioassay if it is determined that the test spectrum maps to then-dimensional space within an acceptable distance from said at least onecentroid in the control model.
 2. The method of claim 1, wherein saiddiluting each serum of the diverse group of sera includes diluting thesera with diluents having a predetermined diluent concentration, andsaid diluting a test serum with a test diluent includes diluting a testserum with a diluent having the same concentration as the diluent usedto dilute each serum of the diverse group of sera.
 3. The method ofclaim 1, wherein said diluting each serum of the diverse group of seraincludes diluting the sera with diluents having a predetermined diluentconcentration, and said diluting a test serum with a test diluentincludes diluting a test serum with a diluent having a differentconcentration than the diluent used to dilute each serum of the diversegroup of sera.
 4. The method of claim 1, further comprising: classifyinga biological state from the spectrum based on a predetermined biologicalstate model.
 5. The method of claim 1, wherein if the test spectrum doesnot map to the n-dimensional space within an acceptable distance fromthe at least one centroid in the control model, and the test diluent isa first diluent, the method further comprising: repeating the steps ofdiluting a test serum, performing, mapping, and certifying for a seconddiluent different from said first diluent.
 6. The method of claim 1,said selecting further comprising: selecting at least two different serafrom a pool of diverse sera, the pool of diverse sera consisting of:sera from healthy males, sera from healthy females, sera from malesafflicted with a disease, sera from females afflicted with a disease,sera from persons of different races, and sera from people of differentages.
 7. The method of claim 1, wherein said generating includes:identifying at least one cluster in common to the sera of the diversegroup of sera and the plurality of different diluents; and selectingonly one cluster as the centroid of the control model.
 8. The method ofclaim 1, wherein the obtaining information includes: obtaininginformation on sera diluted with two different diluents, the diluentsincluding at least acetonitrile and methanol.
 9. The method of claim 1,wherein the test diluent is one of the plurality of different diluents.10. The method of claim 1, wherein the test diluent is not one of theplurality of different diluents.
 11. The method of claim 1, wherein saidplurality of different diluents includes acetonitrile and methanol. 12.The method of claim 1, wherein said diluting each serum of the diversegroup of sera includes creating a plurality of dilutions of each serumwith a diluent at a plurality of concentrations.
 13. The method of claim12, wherein said plurality of concentrations ranges between 1:250 to1:1000.
 14. The method of claim 1, wherein said diluting a test serumincludes diluting a test serum with a known diluent.
 15. The method ofclaim 1, wherein said diluting a test serum includes diluting a testserum with one of the plurality of different diluents used to dilute thediverse group of sera.
 16. The method of claim 1, wherein said dilutinga test serum includes diluting a test serum with a test diluentdifferent than any of the plurality of different diluents used to dilutethe diverse group of sera.
 17. A method of determining whether massspectral data from a test serum is acceptable for analysis in a bioassayfor a biological diagnostic employing a control model generated based onmass spectra obtained from sera analyzed following an electrosprayprocess, the spectra being associated with a plurality of different seraand a plurality of different diluents, the control model including atleast one centroid located in an n-dimensional space defined by n massspectral features included in the control model, comprising: diluting atest serum using a test diluent; ionizing the diluted test serum usingan electrospray process; performing mass spectrometry on the ionizeddiluted test serum to obtain test spectral data associated with the testserum and the test diluent; and mapping the test spectrum to then-dimensional space; and certifying that the test spectrum is acceptablefor analysis in the bioassay if the test spectrum maps to then-dimensional space within an acceptable distance from the centroid. 18.The method of claim 17, wherein the bioassay determines if the testserum exhibits a particular biological state.
 19. The method of claim18, wherein the test diluent is one of acetonitrile and methanol.
 20. Amethod of determining whether mass spectral data from a test serum isacceptable for analysis in a bioassay using sera diluted with a firstdiluent, comprising: providing in an n-dimensional space defined by nmass spectral features a location of at least one centroid associatedwith a first diluent and that distinguishes the first diluent from atleast one second diluent; using an electrospray ionization process,ionizing a test serum diluted with the at least one second diluent togenerate a test mass spectrum; mapping the test mass spectrum to then-dimensional space; and certifying the spectrum for analysis in thebioassay if the spectrum maps to the n-dimensional space within anacceptable distance from the at least one centroid associated with thefirst diluent.
 21. A method of determining whether mass spectral datafrom a test serum using an electrospray process is acceptable foranalysis in a bioassay that generates mass spectral data from a samplethat is diluted by a diluent, comprising: providing a location in ann-dimensional space defined by n mass spectral features of at least onecentroid associated with a preferred diluent concentration andcomposition; providing a location in the n-dimensional space of at leastone test centroid associated with a test sample; comparing the at leastone test centroid to the at least one centroid associated with apreferred diluent to determine the displacement in the n-dimensionalspace of the at least one test centroid; determining a degree of errorbetween the test centroid and the centroid associated with the preferreddiluent; and certifying to a user that the test sample is accepted foranalysis if the degree of error between the test centroid and thecentroid associated with the preferred diluent is acceptable.
 22. Themethod of claim 21, wherein the bioassay determines if the test serumexhibits a particular biological state.
 23. The method of claim 21,wherein the sample is serum.
 24. The method of claim 21, wherein themass spectral data is generated by an electrospray ionization technique.25. A method of determining whether mass spectral data from a test serumusing an electrospray process is acceptable for analysis in a bioassaythat generates mass spectral data from a sample that is diluted by adiluent, comprising: providing a location in an n-dimensional spacedefined by n mass spectral features of at least one centroid associatedwith a preferred diluent concentration and composition; providing alocation in the n-dimensional space of at least one test centroidassociated with a test sample; and determining the displacement in then-dimensional space of the at least one test centroid from the at leastone centroid associated with the preferred diluent; and certifying to auser that the test sample is accepted for analysis if the displacementof the at least one test centroid from the at least one centroidassociated with the preferred diluent is within an acceptable distance.26. The method of claim 25, wherein the bioassay determines if the testserum exhibits a particular biological state.
 27. The method of claim25, wherein the sample is serum.
 28. The method of claim 25, wherein themass spectral data is generated by an electrospray ionization technique.29. A method of determining whether mass spectral data from a test serumusing an electrospray process is acceptable for analysis in a bioassayquality assurance for a biological diagnostic employing a control modelgenerated based on mass spectral features associated with a sample thatincludes serum and a preferred concentration of diluent and compositionof diluent, the control model including at least one centroid located inan n-dimensional space defined by n mass spectral features associatedwith a sample that includes serum and a preferred concentration ofdiluent and composition of diluent and included in the model,comprising: performing mass spectrometry on a test sample that includesserum and a diluent having a concentration and a composition to obtain atest spectrum associated with the test sample; and mapping the testspectrum to the n-dimensional space; and certifying that theconcentration of the diluent and the composition of the diluent areacceptable for the bioassay, if the test spectrum maps to then-dimensional space within an acceptable distance from the at least onecentroid.
 30. The method of claim 29, wherein the bioassay is capable ofdetermining if the test serum exhibits a disease state.